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Am J Physiol Heart Circ Physiol 281: H2518-H2527, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 6, H2518-H2527, December 2001

Mechanism of alpha -adrenergic regulation of expressed hKv4.3 currents

Sunny S. Po*, Richard C. Wu*, George J. Juang, Wei Kong, and Gordon F. Tomaselli

Division of Cardiology, Department of Medicine, and Institute of Molecular Cardiobiology, Johns Hopkins University, Baltimore, Maryland 21205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The transient outward potassium current (Ito) is an important repolarizing current in the mammalian heart. Ito is regulated by adrenergic stimulation; however, the effect of agonists on this current, and consequently the action potential duration and profile, is variable. An important source of the variability is the difference in the channel genes that underlie Ito. There are two subfamilies of candidate genes that are likely to encode Ito in the mammalian heart: Kv4 and Kv1.4; the predominance of either gene is a function of the species, stage of development, and region of the heart. The existence of different isoforms of the Kv4 family (principally Kv4.2 or Kv4.3) further complicates the effect of alpha -adrenergic modulation of cardiac Ito. In the human ventricle, hKv4.3 is the predominant gene underlying Ito. Two splice variants of human Kv4.3 (hKv4.3) are present in the human ventricle; the longer splice variant contains a 19-amino acid insert in the COOH-terminus with a consensus protein kinase C (PKC) site. We used heterologous expression of hKv4.3 splice variants and studies of human ventricular myocytes to demonstrate that alpha -adrenergic modulation of Ito occurs through a PKC signaling pathway and that only the long splice variant (hKv4.3-L) is modulated via this pathway. Only a single hKv4.3-L monomer in the tetrameric Ito channel is required to confer sensitivity to phenylephrine (PE). Mutation of the PKC site in hKv4.3-L eliminates alpha -adrenergic modulation of the hKv4.3-encoded current. The similar, albeit less robust, modulation of human ventricular Ito by PE suggests that hKv4.3-L is expressed in a functional form in the human heart.

potassium channels; adrenergic receptors; heterologous expression; site-directed mutagenesis; protein kinase C; phorbol esters


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CA-INDEPENDENT transient outward potassium current (Ito) is a central component of the repolarization machinery in mammalian ventricular myocytes. The magnitude of Ito is important in controlling the level of the plateau and the overall action potential duration. The expression of Ito is developmentally and pathophysiologically regulated. This complex regulation involves both transcriptional and posttranslational mechanisms. In the human (25), dog (17, 31), and rat (40), Ito density increases with postnatal development. There is evidence for developmental changes in the potassium channel alpha -subunit that underlies Ito (39). Changes in the density of ventricular Ito have been described in a variety of pathological conditions including cardiac hypertrophy and failure (for a review, see Ref. 35).

Functional expression of Ito varies regionally in the adult human (5, 27, 28, 38), canine (3, 23, 24, 36), and rabbit (12) ventricle, generating regional heterogeneity of early repolarization. The heterogeneity of early repolarizing currents is further exaggerated by variations in Ito kinetics in different regions of the human ventricle (28, 38), suggesting the possibility of regional differences in the potassium channel alpha -subunits that underlie Ito (26). In fact, in the rat ventricle, there are regional differences in the expression of Kv4 and Kv1 alpha -subunit mRNA and protein (8, 9, 39). Recently, a calcium-binding subunit that binds to and alters the function of Kv4-encoded currents has been described (2).

The complexities in functional Ito expression have hampered the understanding of the neurohormonal modulation of this current. Adrenergic stimulation is an important modulator of several ionic currents and transporters including Ito. In small mammals, stimulation of alpha -adrenergic receptors prolongs the ventricular action potential and reduces Ito density (4, 6, 10, 13, 32). Much less is known about the adrenergic modulation of Ito in ventricular myocytes of larger mammals and humans. Inconsistencies in the neurohumoral modulation of Ito in native cardiac cells may be the result of species-dependent differences in adrenergic receptors, differences in the potassium channel subunits that underlie Ito, and the presence of other ionic currents that confound the measurement of Ito.

As a first step in understanding the adrenergic modulation of human ventricular Ito, we reconstituted the signaling pathway from receptor to channel in mouse Ltk- fibroblasts. We coexpressed human alpha 1A-adrenergic receptors with human Kv4.3 (hKv4.3), the ion channel gene that is most likely to underlie Ito in the epicardium and midmyocardium of the human ventricle. Similar to the rat (34), there are two splice variants of hKv4.3; the longer variant (hKv4.3-L) contains a 19-amino acid insert in the COOH terminus with a consensus protein kinase C (PKC) phosphorylation site (21). The current generated by expression of hKv4.3-L, but not the short splice variant (hKv4.3-S), is inhibited by phorbol esters and alpha -adrenergic agonists when coexpressed with alpha 1-adrenergic receptors. Coexpression of both hKv4.3-L and hKv4.3-S with the alpha 1-adrenergic receptor was performed to examine the properties of currents produced by heteromultimeric channels and suggests that only a single hKv4.3-L subunit is required to impart alpha 1-adrenergic sensitivity to the channel. Inhibition of hKv4.3-L current is relieved by the presence of PKC inhibitors and by mutation of the spliced-in consensus PKC site.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heterologous expression in Ltk- cells and site-directed mutagenesis. Full-length cDNAs encoding both splice variants of hKv4.3 were subcloned into the pIRES-GFP (Clontech; Palo Alto, CA) plasmid vector for bicistronic expression of the hKv4.3 channel and green fluorescence protein in mouse Ltk- fibroblasts as previously described (21). Transient transfection was performed using either calcium phosphate precipitation or lipofectamine (GIBCO-BRL; Gaithersburg, MD). Cells were cultured in DMEM supplemented with 5% fetal bovine serum and 1% penicillin-streptomycin (GIBCO-BRL) in a 5% CO2 incubator at 37°C for 36-72 h. Cells that emitted green fluorescence were chosen for the patch-clamp experiments. For the alpha -adrenergic stimulation experiments, human alpha 1A- and alpha 1B-adrenergic receptor cDNAs were subcloned into the pIRES-GFP plasmid vector (Clontech) and coexpressed with the hKv4.3 gene in the pcDNA 3.1 plasmid vector (Invitrogen; Carlsbad, CA). Green cells that expressed Ito-like current were considered to express both hKv4.3 current and alpha 1-receptors. To determine the minimal number of hKv4.3-L variants required to impart sensitivity to alpha -adrenergic activation, both hKv4.3-L and hKv4.3-S splice variants were combined in molar ratios of 5 hKv4.3-L:1 hKv4.3-S, 1 hKv4.3-L:1 hKv4.3-S, and 1 hKv4.3-L:5 hKv4.3-S and expressed with the alpha 1A-adrenergic receptor.

The threonine residue at position 503 of hKv4.3-L was changed to alanine by PCR with overlapping mutagenic primers (Quickchange Site-Directed Mutagenesis kit, Stratagene; La Jolla, CA). All mutations were performed in duplicate and confirmed by DNA sequencing of the region surrounding the mutation.

Human ventricular myocyte isolation. Human myocytes were isolated from the midportion of the wall of the left ventricle from explanted hearts by perfusion of a coronary artery with a collagenase-protease solution as previously described (20). The cells were stored at room temperature in Tyrode solution consisting of (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4) supplemented with 10 taurine, 5 sodium pyruvate, and 20 2,3-butanedione monoxime. The concentration of CaCl2 was gradually raised from 100 µM to 2 mM. Only rod-shaped cells with clear striations were selected for electrophysiological study. Ito density in cells isolated from failing hearts was variable, and cells with <1 pA/pF of Ito were excluded from analysis.

Electrophysiology and data analysis. Mouse Ltk- fibroblasts (L-cells) were transferred to the stage of an inverted microscope, superfused with bath solution (see below) at a rate of 1-2 ml/min, and voltage clamped using the whole cell configuration of the patch-clamp technique 24-72 h after transfection. All experiments were performed at room temperature (22-23°C). Currents were recorded using an Axopatch 200A patch-clamp amplifier (Axon Instruments; Foster City, CA) interfaced to a personal computer. Voltage commands were issued and data were collected with custom-written software. Patch electrodes were pulled from borosilicate glass and had 2- to 4-MOmega tip resistances when filled with an internal solution containing (in mM) 110 KCl, 1 MgCl2, 10 HEPES, and 1 EGTA, adjusted to pH 7.2 with KOH to yield a final potassium concentration of 130 mM. When appropriate, 10 µM chelerythrine chloride (Calbiochem; La Jolla, CA) was added to the pipette solution. Cell capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 20-mV hyperpolarizing test pulse from a holding potential of -80 mV.

L-cells were perfused with a standard Tyrode solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose (pH 7.4). Human ventricular myocytes were perfused with the Tyrode solution described above supplemented with 0.3 mM CdCl2 to block calcium currents. Phenylephrine (PE), 4alpha -phorbol 12,13-didecanoate (4alpha -PDD), and phorbol 12-myristate 13-acetate (PMA) solutions (Sigma; St. Louis, MO) were made immediately before each experiment. Full electrophysiological characterization of all channel variants was performed before and after superfusion with 10 µM PE, 100 nM PMA, or 100 nM 4alpha -PDD.

Whole cell currents were elicited by a family of depolarizing voltage steps from a holding potential of -80 mV. In experiments involving myocytes, a 10-ms depolarizing prepulse to 40 mV was applied before depolarizing test pulses to inactivate sodium current. The decay rates were determined by a single exponential fit to the falling phase of the current over a range of voltages from 0 to +80 mV. After induction of steady-state inactivation by 500-ms prepulses from a holding potential of -80 mV, peak outward currents were elicited by a 500-ms test pulse to +50 mV applied every 10 s. Individual steady-state inactivation curves were fitted to the following Boltzmann equation: I/Imax = 1/{1 + exp[(Vp - V1/2)/k']}, where I is current, Imax is the maximal peak current, Vp is the prepulse voltage, V1/2 is the voltage for one-half inactivation, and k' is the slope factor. Recovery from inactivation was assessed by a standard paired pulse protocol: a 500-ms test pulse to +50 mV (P1) was followed by a variable recovery interval at -100 mV and then by a second test pulse to +50 mV (P2). The plot of P2/P1 was fit by a monoexponential function to determine the rates of recovery from inactivation.

Statistical analysis. Comparisons of the splice variants (hKv4.3-L vs. hKv4.3-S) and of mutant and wild-type channels (hKv4.3-L vs. hKv4.3-T503A) were made using an unpaired t-test. All paired data before and after drug exposure were compared using a paired t-test. In all cases, P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of PE on hKv4.3 coexpressed with the alpha 1-adrenergic receptor. The basal whole cell electrophysiological properties of hKv4.3-L and hKv4.3-S expressed in Ltk- cells were not significantly different (21). Coexpression of human alpha 1A-adrenergic receptors with each of the hKv4.3 splice variants did not alter the basal electrophysiology of the currents (Fig. 1 and Table 1). The currents elicited by depolarizing voltage steps activated at -40 to -30 mV exhibited a monotonically increasing current with larger depolarizing voltage steps (Fig. 1, A and B). The time constants for decay of the hKv4.3-L and hKv4.3-S whole cell currents at +60 mV were 68 ± 4 and 64 ± 5 ms, respectively (P = not significant; Table 1). The voltages at one-half maximal availability (V1/2) for each of the hKv4.3 splice variants in the presence of the alpha 1A-receptor were not significantly different (Fig. 1C). Similarly, the recoveries of hKv4.3-L and hKv4.3-S from inactivation were not significantly different from each other in the presence (Fig. 1D) or absence of the alpha 1-receptors (Table 1).


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  		Fig. 1.   Long and short splice variants of human Kv4.3 channels [hKv4.3-L () and hKv4.3-S (), respectively] coexpressed with alpha 1-adrenergic receptors. A: whole cell currents elicited by a family of depolarizing voltage steps from -40 to +60 mV in increments of 10 mV from a holding potential of -80 mV at a frequency of 0.2 Hz for mouse Ltk- cells expressing hKv4.3-L and hKv4.3-S. The current-voltage (V) relationships (B), steady-state availability curves [current (I)/maximal peak current (Imax); C], and recovery from inactivation (D) did not differ between the splice variants when expressed with the alpha 1A-adrenergic receptor. P1 and P2, first and second test pulse, respectively. The basal electrophysiological characteristics of hKv4.3-L and hKv4.3-S currents were not changed by alpha 1A-adrenergic receptor expression [cf Kong et al. (21) and Table 1].


                              
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  		Table 1.   Kv4.3 Splice variants coexpressed with alpha 1A-AR

To examine the coupling of human alpha 1-receptors to hKv4.3 channels, we exposed cells to 10 µM PE. The peak current elicited by a voltage step from -80 to +50 mV was inhibited within 3 min and decreased by ~50% within 15 min of exposure to PE in cells expressing the alpha 1A-adrenergic receptor and hKv4-3-L (Fig. 2, A, C, and E). In contrast, PE had no significant effect on hKv4.3-S (Fig. 2, B and D), and the small reduction of peak current was similar to the rundown observed for expressed hKv4.3 channels in the absence of PE. In the absence of the human alpha 1-receptors, PE had no significant effect on hKv4.3-L or hKv4.3-S, suggesting the absence of alpha 1-adrenergic receptors in mouse Ltk- cells or failure of endogenous receptors to couple to the expressed channels (Fig. 2F). The reduction in hKv4.3-L peak current may be due to a change in the channel density or the result of an alteration in voltage dependence or kinetics induced by PE.


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  		Fig. 2.   Phenylephrine (PE) inhibits hKv4.3-L. A and B: peak currents elicited by voltage pulses from -80 to +50 mV at a repetition interval of 20 s through expressed hKv4.3-L (A) and hKv4.3-S (B) without (open circle  and ) and with the application of 10 µM PE ( and ). Insets, currents before and 15 min after (*) infusion of PE for hKv4.3-L and hKv4.3-S. C: current-voltage relationship of hKv4.3-L before (open circle ) and after () exposure to 10 µM PE. D: current-voltage relationship of hKv4.3-S in the presence () and absence () of PE. E: bar plot of the current before and after 15 min exposure to PE. hKv4.3-L was reduced by 54 ± 7% (P < 0.05). There was no significant change in the peak hKv4.3-S current. F: in the absence of the alpha 1A-adrenergic receptor, PE had no effect on either hKv4.3-L () or hKv4.3-S (data not shown); open circle , hKv4.3-L channels without PE treatment.

PE produced no significant change in the voltage dependence of the current-voltage relationship of hKv4.3-L (Fig. 2C). The steady-state availability was determined using 500-ms preconditioning pulses to voltages from -100 to +0 mV in increments of 10 mV. The availability curve of hKv4.3-L was significantly shifted in the hyperpolarizing direction by PE (V1/2 = -43.6 ± 0.6 mV in control vs. -53.0 ± 0.5 mV with PE, n = 6, P < 0.05) without a significant change in the slope of the relationship (6.2 ± 0.4 mV in control vs. 7.6 ± 0.4 mV with PE, n = 6; Fig. 3A). PE did not significantly alter the steady-state availability relationship of hKv4.3-S (V1/2 = -44.3 ± 1.2 mV in control vs. -49.3 ± 2.6 mV with PE cf 8.3 ± 0.6 mV in control vs. 8.7 ± 0.6 mV with PE, n = 5; Fig. 3B).


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  		Fig. 3.   PE alters the availability and recovery of hKv4.3-L. Steady-state availability was measured with 500-ms conditioning pulses from -100 to 0 mV followed by a test pulse to +50 mV for hKv4.3-L (A) and hKv4.3-S (B) before (open circle  and , solid lines) and after ( and , dashed-dotted lines) exposure to 10 µM PE. The voltage for one-half maximal activation (V1/2) and slope factors were determined by Boltzmann fits to the data, as described in MATERIALS AND METHODS. PE significantly shifted the V1/2 of hKv4.3-L but not hKv4.3-S in the hyperpolarizing direction (Table 1). A two-pulse protocol was used to measure recovery from inactivation at -100 mV for hKv4.3-L (C) and hKv4.3-S (D). The data were fit by single exponentials and the time constants for the fits are given in Table 1.

The recovery kinetics of both channels were studied at -100 mV after a depolarizing voltage step to +50 mV for 500 ms. The time course of recovery was assayed by plotting the ratio of the peak current amplitudes (P1/P2) over a range of recovery intervals. The time course of recovery from inactivation of hKv4.3-L was significantly slowed by PE (89 ± 5 ms in control vs. 154 ± 10 ms with PE, n = 5, P < 0.05; Fig. 3C), whereas that of hKv4.3-S was not significantly altered (95 ± 9 ms in control vs. 103 ± 17 ms with PE, n = 4; Fig. 3D).

Effects of phorbol esters, PKC inhibitors, and phosphorylation site mutation. We examined whether the isoform-specific modulation of hKv4.3-L by PE was due to phosphorylation of the channel using several complementary approaches. Activation of PKC by phorbol esters reproduced most of the effects of alpha 1-adrenergic stimulation on hKv4.3-L. hKv4.3-S was unaffected by PMA (Fig. 4B) and 4alpha -PDD (data not shown). In contrast, the peak hKv.43-L current was significantly reduced by 100 nM PMA (46 ± 8%, P < 0.05; Figs. 4, A, C, and D). Similar to PE, PMA shifted the availability curve of hKv4.3-L in the hyperpolarizing direction (-41.7 ± 0.6 mV in control vs. -57.6 ± 1.1 mV with PE, P < 0.05) with a modest but significant increase in the slope factor (6.8 ± 0.5 mV in control vs. 8.9 ± 1.0 mV with PE, P < 0.05; Fig. 4E). The time course of recovery from inactivation of hKv4.3-L at -100 mV was not significantly prolonged by PMA (83.5 ± 4.9 ms in control vs. 95.6 ± 4.3 ms with PMA; Fig. 4F).


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  		Fig. 4.   Phorbol 12-myristate 13-acetate (PMA) mimics the effect of PE on expressed hKv4.3-L. Currents were elicited by depolarizing voltage steps from -80 to +50 mV for 500 ms in mouse Ltk- cells expressing hKv4.3-L before and after (*) exposure to 100 nM PMA (A) and the inactive phorbol ester 4alpha -phorbol 12,13-didecanoate (4alpha -PDD; B). C: current-voltage relationship of hKv4.3-L before (open circle  and solid lines) and after 20-min exposure to PMA ( and dashed-dotted lines) or 4alpha -PDD (). PMA produced a voltage-independent reduction of the current, and there was no effect of 4alpha -PDD. D: bar plot of the peak currents elicited by steps to +50 mV after 15-min exposure to PMA and 4alpha -PDD normalized to the peak before drug exposure. E: PMA shifted the steady-state availability curve of hKv4.3-L by 15.9 mV in the hyperpolarizing direction and increased the slope factor. 4alpha -PDD did not alter the availability curve of hKv4.3-L. F: PMA slowed recovery from inactivation, but the change did not reach statistical significance. 4alpha -PDD had no effect on the recovery of hKv4.3-L.

To further explore the role of PKC-mediated phosphorylation in the modulation of hKv4.3-L, we examined the effect of inhibitors of PKC on alpha -agonist-induced inhibition of the current. Chelerythrine (1 µM) in the patch pipette eliminated the PE-induced reduction of expressed hKv4.3-L. Selective inhibition of PKC also eliminated the PE-induced hyperpolarizing shift of the hKv4.3-L availability curve and the slowing of recovery from inactivation (Table 1).

Activation of PKC by alpha -adrenergic stimulation may phosphorylate a number of effector proteins. To determine whether hKv4.3-L phosphorylation by PKC mediates the change in the expressed current, we mutated the consensus phosphorylation site in the splice insert. The threonine residue at position 503 was mutated to alanine (hKv4.3-T503A) to eliminate the PKC phosphorylation site of the form RXXT*XK in the 19-amino acid insert of hKv4.3-L. hKv4.3-T503A had basal electrophysiological properties that were unchanged from the wild-type channel (Fig. 5A and Table 1). When hKv4.3-T503A was coexpressed with alpha 1A-receptors, the peak current was unaffected by application of PE (Fig. 5A) or PMA (data not shown). Similarly, the availability curve of hKv4.3-T503A was unaffected by PE (Fig. 5B). Recovery from inactivation of the mutant channel was identical in the presence and absence of PE (Fig. 5C).


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  		Fig. 5.   hKv4.3-T503A mutant is insensitive to alpha -adrenergic stimulation. A: exposure of the wild-type hKv4.3-S () and hKv4.3-T503A mutant (triangle ) coexpressed with human alpha 1A-receptors to 10 µM PE; the format is similar to that of Fig. 2A. Inset, currents through the mutant channels elicited by a depolarizing voltage step from -80 to +50 mV before and after (*) 15-min exposure to PE. B and C: steady-state availability (B) and recovery from inactivation (C) of the hKv4.3-T503A mutant before (triangle  and solid lines) and after (black-triangle and dashed-dotted lines) exposure to PE. The mutant current was unaffected by PE and PMA (data not shown).

Coexpression of hKv4.3-S and hKv4.3-L with the alpha 1-adrenergic receptor was performed to examine the properties of currents produced by heteromultimeric channels. To determine whether a single hKv4.3-L subunit is sufficient to render the channel susceptible to alpha 1-adrenergic modulation, we coexpressed hKv4.3-S and hKv4.3-L subunits in varying ratios (1:5, 1:1, and 5:1). If a single subunit is sufficient, 17% of the PE-induced shift in steady-state inactivation or a reduction of peak homotetrameric hKv4.3-L current would be expected when the subunits are expressed in 5:1 (hKv4.3-S to hKv4.3-L) ratio. The current amplitudes of the heteromultimeric channels were reduced for each mixture in the presence of PE. The fractional current reduction for each mixture was normalized to the fractional reduction of homotetrameric hKv4.3-L currents by PE. Current through hybrid channels expressed with a 5:1 molar ratio of hKv4.3-S to hKv4.3-L was reduced to 23 ± 5% of the PE-induced reduction of hKv4.3-L current. When coexpressed in a 1:1 ratio, peak current reduction was 64 ± 3% of that induced by PE treatment of hKv4.3-L channels. The effect of PE on peak current when hKv4.3-L was present in a fivefold greater molar ratio than hKv4.3-S was not significantly different from the effect on hKv4.3-L homotetrameric channels (Fig. 6). Similarly, a measurable shift with the steady-state inactivation curve was present with the 1:5 molar ratio of hKv4.3-L to hKv4.3-S, and the effect was nearly maximal when the splice variants were expressed in equimolar ratios (Table 2).


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  		Fig. 6.   Coexpression of hKv4.3-L and hKv4.3-S with alpha 1-adrenergic receptors. Reduction of peak current (Ipeak) through heteromultimeric channels was normalized to the reduction of homotetrameric hKv4.3-L current in the presence of PE. Insets, reduction of peak current through channels comprising 5:1 (a), 1:1 (b), and 1:5 (c) ratios of hKv4.3-S to hKv4.3-L, respectively. The effect of PE observed when channels are expressed in a 1:5 molar ratio of hKv4.3-S to hKv4.3-L was not different from hKv4.3-L homotetrameric channels. Solid line, model in which a single hKv4.3-L subunit in the channel complex is sufficient to impart PE sensitivity; dotted line, model in which hKv4.3-L homotetrameric channels are necessary for PE effect.


                              
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  		Table 2.   Kv4.3 Heteromultimers coexpressed with alpha 1A-AR

We then examined the effect of PE on native Ito in human cardiac ventricular myocytes. Cells (n = 8) isolated from the mid left ventricular wall of five explanted hearts (2 ischemic and 2 dilated cardiomyopathic) were studied. The cells selected exhibited a robust Ito (Fig. 7A) that shifted in the depolarizing direction compared with expressed Kv4.3 due to the presence of CdCl2 in the recording solution (1). The peak cardiac Ito was decreased by 28 ± 18% at + 60 mV in the presence of 10 µM PE (Fig. 7, A-C). There was a small hyperpolarizing shift of the availability curve and slowing of the recovery from inactivation at -100 mV; however, no change reached statistical significance.


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  		Fig. 7.   Effect of PE on human cardiac transient outward potassium current (Ito). Ito was recorded from human myocytes isolated from the mid left ventricular wall before (A) and after (B) the application of 10 µM PE and 1 µM propranolol. The currents were elicited by test pulses from -30 to +80 mV in increments of 10 mV from a holding potential of -80 mV. Dotted line, zero current. The scale bars are 5 pA/pF and 200 ms. C: current-voltage relationship of Ito before (open circle ) and after PE-propranolol (; n = 5 hearts and n = 8 cells). D: PE-propranolol produced a small but statistically significant hyperpolarizing shift of the steady-state availability curve (control: open circle  and solid lines, V1/2 = -17.7 ± 0.5 cf 7.7 ± 0.5 mV; PE:  and dashed-dotted lines, V1/2 -24.5 ± 0.4 cf 7.6 ± 0.4 mV, P = 0.006). E: recovery from inactivation before (open circle  and solid lines) and after PE-propranolol ( and dashed-dotted lines). The data are plotted on a logarithmic scale; the fast recovery time constants, representing >95% of current recovery, were 19.5 ± 5 ms before (open circle ) and 28.9 ± 10 ms after PE (, P = not significant).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Repolarization abnormalities play a critical role in the genesis of both congenital and acquired cardiac arrhythmias (e.g., Ref. 33). Ito is an important current in early repolarization of the human ventricle and its density is highly regulated (5, 7, 14, 16, 20, 22, 25-28, 37, 38). The role and indeed the effect of alpha -adrenergic stimulation on Ito are controversial (11, 15). Among the possible causes of the diversity of effects of alpha -adrenergic stimulation on cardiac Ito are the molecular components that constitute this current.

There are several candidate genes that may encode cardiac Ito in large mammals like the dog and human; Kv4.3 (9, 18-20), possibly in combination with KChiP2 (2), is the leading candidate, although Kv1.4 may also play a role, particularly in the endocardium (18, 20, 26). We cloned two splice variants of the human Kv4.3 gene from the human heart that differ from each other, with the hKv4.3-L variant having a 19-amino acid insert in the COOH terminus near the sixth transmembrane domain (21, 34). This insert contains a consensus sequence for PKC phosphorylation. Both hKv4.3-S and hKv4.3-L encode channels that conduct currents that resemble human ventricular Ito, with no significant differences between the two splice variants in their basal electrophysiological properties (21).

Previous studies of alpha -adrenergic modulation of cardiac Ito in different species have yielded variable results. Although reduction of Ito density is a generally consistent observation, the signal transduction pathway involved is less certain. PKC is a common effector of a variety of cell surface receptors such as endothelin, angiotensin, and alpha -adrenergic receptors. However, it is unclear whether the downregulation of Ito by alpha -adrenergic stimulation is mediated through activation of PKC (4, 6, 29, 30). We sought to understand the alpha -adrenergic regulation of the hKv4.3 splice variants and correlate these findings with alpha -agonist-mediated effects on native human ventricular Ito.

Coexpression of human alpha 1-adrenergic receptors with hKv4.3 reconstitutes a transient outward current that is regulated by alpha -adrenergic agonists. The peak current amplitude of hKv4.3-L, but not hKv4.3-S, was decreased by application of PE to cells expressing the channel subunit and either human alpha 1A- or alpha 1B-adrenergic (data not shown) receptors. Support for the hypothesis of PKC-mediated alpha -adrenergic inhibition of Ito comes from several independent lines of evidence. The effect of alpha 1-adrenergic receptor stimulation is mimicked by diacetyl glycerol (DAG) analogs. Suppression of hKv4.3-L current by alpha -adrenergic agonists is blocked by the PKC inhibitor chelerythrine and by mutation of a consensus PKC phosphorylation site (hKv4.3-T503A) in the COOH terminus of this splice variant. Thus inhibition of hKv4.3-encoded Ito by alpha -adrenergic stimulation is mediated via PKC phosphorylation of the channel protein at a site in the COOH terminus unique to hKv4.3-L. Downregulation of Ito by alpha -adrenergic stimulation is in part due to a change in the voltage dependence of availability, but the reduction in peak current exceeds that predicted by the shift in the steady-state inactivation curve. On the basis of the magnitude of the steady-state inactivation gating shift and reduction in the peak current when hKv4.3-L and hKv4.3-S were coexpressed in differing molar ratios, we suggest that only a single hKv4.3-L subunit in a tetrameric channel complex is required to impart sensitivity to alpha -adrenergic stimulation (Fig. 6).

hKv4.3-L expressed without alpha -adrenergic receptors is not affected by application of PE, but the peak current is reduced by phorbol esters. If endogenous alpha -receptors are present on the surface of mouse Ltk- cells, they are not effectively coupled to the expressed channels. hKv4.3-L is modified by DAG in a manner similar to that observed with PE when the channel is coexpressed with human alpha 1A-adrenergic receptors. Thus the signaling cascade downstream of the receptor is intact in Ltk- cells and involves activation of PKC.

Ventricular myocytes isolated from human hearts at the time of cardiac transplantation express both splice variants of hKv4.3 by Western blotting (18). Ito in human left ventricular myocytes is inhibited by PE in the presence of beta -adrenergic receptor blockers, and the effects are qualitatively similar to that of expressed hKv4.3-L but do not reach statistical significance. The similarity of the biophysical features of expressed hKv4.3 and native human ventricular Ito suggests that this potassium channel alpha -subunit, at least in part, encodes Ito. The inhibition of Ito by PE further suggests that hKv4.3-L is expressed in a functional form in the human ventricle and accounts for a component of the alpha -adrenergic receptor-mediated modulation of native cardiac Ito. The smaller effect of PE on native Ito may be due to a number factors. Such factors include the presence of accessory subunits, a more predominant functional role of hKv4.3-S in comprising native Ito, or alterations in the coupling between alpha -adrenergic receptors and the channel in cells isolated from failing human ventricles.

Limitations. This study focused on the role of alpha -adrenergic stimulation on the function of expressed hKv4.3 channel. It does not consider the possible role of accessory subunits such as KChIP2 (2). To the extent that any other Kv subunit (e.g., Kv1.4) underlies Ito, the effects on expressed hKv4.3 may not completely recapitulate the effects of alpha -adrenergic stimulation on native cardiac Ito. However, we observed very little (<5%) slowly recovering current in human ventricular myocytes, suggesting a negligible contribution to Ito by Kv1.4 in cells isolated from the mid left ventricular wall. As with any study on a reconstituted signaling system, the effect of alpha -adrenergic stimulation on hKv4.3 was examined in isolation without the influence of other potential modulators of Ito unique to the heart. The effects of alpha -adrenergic stimulation studied here are acute; chronic exposure to elevated levels of catecholamines, such as in heart failure, is likely to have distinct effects on adrenergic receptors, coupling G proteins, and effectors such as ion channels.


    ACKNOWLEDGEMENTS

We thank Ailsa Mendez-Fitzwilliam for technical support.


    FOOTNOTES

* Authors contributed equally to this study.

This work was supported by National Heart, Lung, and Blood Institute Grants P50 HL-52307 (to G. F. Tomaselli) and T32 HL-07227 (to S. S. Po) and American Heart Association (AHA) Maryland Affiliate Grant-in-Aid S98711M (to G. F. Tomaselli), and by an AHA Maryland Affiliate fellowship grant (to G. J. Juang and W. Kong) and a Stanley J. Sarnoff Endowment Scholars Grant (to R. C. Wu).

Address for reprint requests and other correspondence: G. F. Tomaselli, 844 Ross Bldg., Johns Hopkins Univ. School of Medicine, Baltimore, MD 21205 (E-mail: gtomasel{at}jhmi.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10 May 2001; accepted in final form 29 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(6):H2518-H2527
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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